miR-200a Regulates Epithelial-Mesenchymal to Stem-like Transition via ZEB2 and β-Catenin Signaling

The emerging concept of generating cancer stem cells from epithelial-mesenchymal transition has attracted great interest; however, the factors and molecular mechanisms that govern this putative tumor-initiating process remain largely elusive. We report here that miR-200a not only regulates epithelial-mesenchymal transition but also stem-like transition in nasopharyngeal carcinoma cells. We first showed that stable knockdown of miR-200a promotes the transition of epithelium-like CNE-1 cells to the mesenchymal phenotype. More importantly, it also induced several stem cell-like traits, including CD133⁺ side population, sphere formation capacity, in vivo tumorigenicity in nude mice, and stem cell marker expression. Consistently, stable overexpression of miR-200a switched mesenchyme-like C666-1 cells to the epithelial state, accompanied by a significant reduction of stem-like cell features. Furthermore, in vitro differentiation of the C666-1 tumor sphere resulted in diminished stem-like cell population and miR-200a induction. To investigate the molecular mechanism, we demonstrated that miR-200a controls epithelial-mesenchymal transition by targeting ZEB2, although it regulates the stem-like transition differentially and specifically by β-catenin signaling. Our findings reveal for the first time the function of miR-200a in shifting nasopharyngeal carcinoma cell states via a reversible process coined as epithelial-mesenchymal to stem-like transition through differential and specific mechanisms.     

Role of p38γ MAPK in regulation of EMT and cancer stem cells

p38γ is a member of p38 MAPK family which contains four isoforms p38α, p38β, p38γ, and p38δ. p38γ MAPK has unique function and is less investigated. Recent studies revealed that p38γ MAPK may be involved in tumorigenesis and cancer aggressiveness. However, the underlying cellular/molecular mechanisms remain unclear. Epithelial-mesenchymal transition (EMT) is a process that epithelial cancer cells transform to facilitate the loss of epithelial features and gain of mesenchymal phenotype. EMT promotes cancer cell progression and metastasis, and is involved in the regulation of cancer stem cells (CSCs) which have self-renewal capacity and are resistant to chemotherapy and target therapy. We showed that p38γ MAPK significantly increased EMT in breast cancer cells; over-expression of p38γ MAPK enhanced EMT while its down-regulation inhibited EMT. Meanwhile, p38γ MAPK augmented CSC population while knock down of p38γ MAPK decreased CSC ratio in breast cancer cells. MicroRNA-200b (miR-200b) was down-stream of p38γ MAPK and inhibited by p38γ MAPK; miR-200b mimics blocked p38γ MAPK-induced EMT while miR-200b inhibitors promoted EMT. p38γ MAPK regulated miR-200b through inhibiting GATA3. p38γ MAPK induced GATA3 ubiquitination, leading to its proteasome-dependent degradation. Suz12, a Polycomb group protein, was down-stream of miR-200b and involved in miR-200b regulation of EMT. Thus, our study established an important role of p38γ MAPK in EMT and identified a novel signaling pathway for p38γ MAPK–mediated tumor promotion.     

Over-expression of FoxM1 leads to epithelial-mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial-to-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotypes are highly inter-related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self-renewal capacity is poorly understood. Therefore, we established FoxM1 over-expressing pancreatic cancer (AsPC-1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over-expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E-cadherin, and vimentin, which is consistent with increased sphere-forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over-expression of FoxM1 led to decreased expression of miRNAs (let-7a, let-7b, let-7c, miR-200b, and miR-200c); however, re-expression of miR-200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E-cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo-preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over-expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR-200b and these processes, could be easily attenuated by genistein.     

miR-375 Mediated Acquired Chemo-Resistance in Cervical Cancer by Facilitating EMT

Acquired chemo-resistance is one of the key causal factors in cancer death. Emerging evidences suggest that miRNA and epithelial–mesenchymal transition play critical roles in the chemo-resistance in cancers. Here, we showed the association of paclitaxel-resistance with miR-375 over-expression and epithelial–mesenchymal transition inducement in cervical cancer. Using different cervical cancer cell models, we found that paclitaxel transiently induced up-regulation of miR-375 expression, proliferation inhibition, transition from epithelial to mesenchymal phenotype, and consequently impaired paclitaxel sensitivity. Forced over-expression of miR-375 may suppress Ecadherin expression by a directly targeting pathway, which led to paclitaxel resistance. Contrarily, re-expression of Ecadherin partly reversed epithelial–mesenchymal transition phenotype and miR-375 induced paclitaxel-resistance. Our findings suggest that paclitaxel-induced miR-375 over-expression facilitates epithelial–mesenchymal transition process via directly targeting Ecadherin, proliferation inhibition, and consequently results in chemo-resistance in cervical cancer cells. A reversion of miR-375 or Ecadherin expression may be a novel therapeutic approach for overcoming chemo-resistance in cervical cancer.     

Downregulation of miR-200a Induces EMT Phenotypes and CSC-like Signatures through Targeting the β-catenin Pathway in Hepatic Oval Cells

Hepatocellular carcinoma (HCC) can be derived from malignant transformed adult hepatic progenitor cells. However, the regulatory factors and molecular mechanisms underlying the process are not well defined. Our previous microRNA (miRNA) microarray analysis revealed a significant decrease of miR-200a level in F344 rat HCC side population (SP) fraction cells versus their normal counterparts. In the present study, we further investigated the effect of miR-200a on hepatic oval cell (HOC) phenotypes. We first confirmed downregulated miR-200a levels in rat hepatoma cells compared with WB-F344 cells. Next, by lentivirus-mediated loss-of-function studies, we showed that stable knockdown of miR-200a confers a mesenchymal phenotype to WB-F344 cells, including an elongated cell morphology, enhanced cell migration ability and expression of epithelial mesenchymal transition (EMT)-representative markers. Concomitantly, several cancer stem cell (CSC)-like traits appeared in these cells, which exhibit enhanced spheroid-forming capacity, express putative hepatic CSC markers and display superior resistance to chemotherapeutic drugs in vitro. Furthermore, bioinformatics analysis, luciferase assays and western blot analysis identified β-catenin (CTNNB1) as a direct and functional target of miR-200a. Knockdown of miR-200a partially activated Wnt/β-catenin signaling, and silencing of β-catenin functionally attenuated anti-miR-200a effects in vitro in WB-F344 cells. At length, in vivo xenograft assay demonstrated the acquisition of tumorigenicity of WB-F344 cells after miR-200a siliencing. Collectively, our findings indicate that miR-200a may function as an important regulatory factor in neoplastic transition of HOCs by targeting the β-catenin pathway.     

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation

The miR-200 family consists of five members expressed as two clusters: miR-200c/141 cluster and miR-200b/200a/429 cluster. In the mammary gland, miR-200s maintain epithelial identity by decreasing the expression of mesenchymal markers leading to high expression of epithelial markers. While the loss of miR-200s is associated with breast cancer growth and metastasis the impact of miR-200 expression on mammary tumor initiation has not been investigated. Using mammary specific expression of the miR-200b/200a/429 cluster in transgenic mice, we found that elevated expression miR-200s could almost completely prevent mammary tumor development. Only 1 of 16 MTB-IGFIRba429 transgenic mice (expressing both the IGF-IR and miR-200b/200a/429 transgenes) developed a mammary tumor while 100% of MTB-IGFIR transgenic mice (expressing only the IGF-IR transgene) developed mammary tumors. RNA sequencing, qRT-PCR, and immunohistochemistry of mammary tissue from 55-day old mice found Spp1, Saa1, and Saa2 to be elevated in mammary tumors and inhibited by miR-200b/200a/429 overexpression. This study suggests that miR-200s could be used as a preventative strategy to protect women from developing breast cancer. One concern with this approach is the potential negative impact miR-200 overexpression may have on mammary function. However, transgenic overexpression of miR-200s, on their own, did not significantly impact mammary ductal development indicating the miR-200 overexpression should not significantly impact mammary function. Thus, this study provides the initial foundation for using miR-200s for breast cancer prevention and additional studies should be performed to identify strategies for increasing mammary miR-200 expression and determine whether miR-200s can prevent mammary tumor initiation by other genetic alterations.     

Transgenic overexpression of the miR-200b/200a/429 cluster inhibits mammary tumor initiation

The miR-200 family consists of five members expressed as two clusters: miR-200c/141 cluster and miR-200b/200a/429 cluster. In the mammary gland, miR-200s maintain epithelial identity by decreasing the expression of mesenchymal markers leading to high expression of epithelial markers. While the loss of miR-200s is associated with breast cancer growth and metastasis the impact of miR-200 expression on mammary tumor initiation has not been investigated. Using mammary specific expression of the miR-200b/200a/429 cluster in transgenic mice, we found that elevated expression miR-200s could almost completely prevent mammary tumor development. Only 1 of 16 MTB-IGFIRba429 transgenic mice (expressing both the IGF-IR and miR-200b/200a/429 transgenes) developed a mammary tumor while 100% of MTB-IGFIR transgenic mice (expressing only the IGF-IR transgene) developed mammary tumors. RNA sequencing, qRT-PCR, and immunohistochemistry of mammary tissue from 55-day old mice found Spp1, Saa1, and Saa2 to be elevated in mammary tumors and inhibited by miR-200b/200a/429 overexpression. This study suggests that miR-200s could be used as a preventative strategy to protect women from developing breast cancer. One concern with this approach is the potential negative impact miR-200 overexpression may have on mammary function. However, transgenic overexpression of miR-200s, on their own, did not significantly impact mammary ductal development indicating the miR-200 overexpression should not significantly impact mammary function. Thus, this study provides the initial foundation for using miR-200s for breast cancer prevention and additional studies should be performed to identify strategies for increasing mammary miR-200 expression and determine whether miR-200s can prevent mammary tumor initiation by other genetic alterations.     

The “good-cop bad-cop” TGF-beta role in breast cancer modulated by non-coding RNAs

BackgroundLack of early diagnosis methods and the development of drug resistance are among the main reasons for increased mortality rates within breast cancer patients. These two aspects are governed by specific pro-carcinogenic modifications, where TGBβ-induced EMT is one of the leading actors. Endowment of the epithelial cells with mesenchymal characteristics allows them to migrate and invade secondary tissues in order to form malignant sites and also confers chemoresistance. TGFβ which role switches from the tumor suppressor cytokine to the oncogenic one favoring the tumor microenvironment regulates this process.Scope of reviewThis review aims to comprehensively present the updated TGFβ-induced EMT in breast cancer, including the regulatory role of the non-coding RNAs with focus on the miR-200 family and newly discovered lncRNAs such as HOTAIRM1. Additionally, a new phenotype, P-EMT, also modulated by miR-200 and miR-34 families that form complex feedback loops with TGFβ, SNAI1 and ZEB1/2 is presented under an updated form.Major conclusionsThe hallmarks of EMT are becoming increasingly associated with aggressive forms of breast cancer and low survival rates among patients. Considering that this phenotypical switch can trigger drug resistance, invasion and metastasis, inhibition of EMT could represent an important milestone in mammary cancer treatment.General significanceThe present review assembles the most recent data regarding TGFβ induced EMT, including the input of non-coding RNAs, contributing to the possible development of new targeted treatment strategies for cancer patients.     

miR‐200c enhances radiosensitivity of human breast cancer cells

Due to the intrinsic resistance of many tumors to radiotherapy, current methods to improve the survival of cancer patients largely depend on increasing tumor radiosensitivity. It is well‐known that miR‐200c inhibits epithelial–mesenchymal transition (EMT), and enhances cancer cell chemosensitivity. We sought to clarify the effects of miR‐200c on the radiosensitization of human breast cancer cells. In this study, we found that low levels of miR‐200c expression correlated with radiotolerance in breast cancer cells. miR‐200c overexpression could increase radiosensitivity in breast cancer cells by inhibiting cell proliferation, and by increasing apoptosis and DNA double‐strand breaks. Additionally, we found that miR‐200c directly targeted TANK‐binding kinase 1 (TBK1). However, overexpression of TBK1 partially rescued miR‐200c mediated apoptosis induced by ionizing radiation. In summary, miR‐200c can be a potential target for enhancing the effect of radiation treatment on breast cancer cells. J. Cell. Biochem. 114: 606–615, 2013. © 2012 Wiley Periodicals, Inc.     

Detecting and targeting mesenchymal-like subpopulations within squamous cell carcinomas

Curative eradication of all cells within carcinomas is seldom achievable with chemotherapy alone. This limitation may be partially attributable to tumor cell subpopulations with intrinsic resistance to current drugs. Within squamous cell carcinoma (SCC) cell lines, we previously characterized a subpopulation of mesenchymal-like cells displaying phenotypic plasticity and increased resistance to both cytotoxic and targeted agents. These mesenchymal-like (Ecad-lo) cells are separable from epithelial-like (Ecad-hi) cells based on loss of surface E-cadherin and expression of vimentin. Despite their long-term plasticity, both Ecad-lo and Ecad-hi subsets in short-term culture maintained nearly uniform phenotypes after purification. This stability allowed testing of segregated subpopulations for relative sensitivity to the cytotoxic agent cisplatin in comparison to salinomycin, a compound with reported activity against CD44⁺CD24⁻ stem-like cells in breast carcinomas. Salinomycin showed comparable efficacy against both Ecad-hi and Ecad-lo cells in contrast to cisplatin, which selectively depleted Ecad-hi cells. An in vivo correlate of these mesenchymal-like Ecad-lo cells was identified by immunohistochemical detection of vimentin-positive malignant subsets across a part of direct tumor xenografts (DTXs) of advanced stage SCC patient samples. Cisplatin treatment of mice with established DTXs caused enrichment of vimentin-positive malignant cells in residual tumors, but salinomycin depleted the same subpopulation. These results demonstrate that mesenchymal-like SCC cells, which resist current chemotherapies, respond to a treatment strategy developed against a stem-like subset in breast carcinoma. Further, they provide evidence of mesenchymal-like subsets being well-represented across advanced stage SCCs, suggesting that intrinsic drug resistance in this subpopulation has high clinical relevance.Key words: EMT, squamous cell carcinoma, head and neck cancer, esophageal cancer, chemotherapy resistance, salinomycin, tumor heterogeneity     

Real-Time Imaging of the Epithelial-Mesenchymal Transition Using microRNA-200a Sequence-Based Molecular Beacon-Conjugated Magnetic Nanoparticles

The epithelial-mesenchymal transition (EMT) plays important roles in tumor progression to metastasis. Thus, the development of an imaging probe that can monitor transient periods of the EMT process in live cells is required for a better understanding of metastatic process. Inspired by the fact that the mRNA expression levels of zinc finger E-box-binding homeobox 1 (ZEB1) increase when cells adopt mesenchyme characteristics and that microRNA-200a (miR-200a) can bind to ZEB1 mRNA, we conjugated molecular beacon (MB) mimicking mature miR-200a to magnetic nanoparticles (miR-200a-MB-MNPs) and devised an imaging method to observe transitional changes in the cells during EMT. Transforming growth factor-β1 treated epithelial cells and breast cancer cell lines representing both epithelial and mesenchymal phenotypes were used for the validation of miR-200a-MB-MNPs as an EMT imaging probe. The real-time imaging of live cells acquired with the induction of EMT revealed an increase in fluorescence signals by miR-200a-MB-MNPs, cell morphology alterations, and the loss of cell-cell adhesion. Our results suggest that miR-200a-MB-MNPs can be used as an imaging probe for the real-time monitoring of the EMT process in live cells.     

A Pituitary Homeobox 2 (Pitx2):microRNA-200a-3p:β-catenin Pathway Converts Mesenchymal Cells to Amelogenin-expressing Dental Epithelial Cells

Pitx2, Wnt/β-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:β-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and β-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and β-catenin expression. Pitx2 and β-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.